The recently-discovered Type VI Secretion System (T6SS), found in many Gram-negative bacterial pathogens, is a protein secretion system that delivers effectors with either virulence or anti-bacterial activities to mediate infection or inter bacterial competition, respectively. Although much has been learned about the T6SS and several effector families have been discovered in the past several years, many T6SSs in various bacteria still lack identifiable effectors and their activities are still unknown. I recently discoered a new wide- spread class of T6SS effectors found in diverse bacteria. These effectors have polymorphic C-terminal toxic domains, many of which have no known function, yet they share an N-terminal region that contains a conserved motif named MIX (Marker for type sIX effectors). Notably, the mechanism of secretion of these MIX- effectors through the T6SS, as well as the function of the MIX motif, remain unknown. In this proposal, I aim at addressing these questions in the following aims. Aims 1: Determine the mechanism of secretion for MIX- effectors. I will use biochemical and genetic approaches to identify what core-component of the T6SS is used by MIX-effectors to mediate their secretion, as well as determine whether the MIX motif serves as a T6SS secretion signal. Aim 2: Identify activity of a common toxin domain associated with MIX-effectors. As many MIX-effectors have no identifiable or known toxic domains, finding their activities will shed light on bacterial virulence and anti-bacterial mechanisms and targets. I will determine the activity of the Duf2235 domain that is common in MIX-effectors as well as in other classes of T6SS effectors. The long-term goal of this proposal is to utilize the anti-bacterial properties of T6SSs, and specifically of MIX-effectors, to develop a new strategy to combat bacterial infections. My observations suggest that MIX-effectors are interchangeable between bacterial species. Therefore, I propose to utilize MIX-effectors with diverse activities in Aim 3: Test whether T6SS MIX- effectors can be manipulated and used as anti-bacterial agents. I will determine whether MIX-effectors can be expressed in heterologous bacterial species and used to widen the range of T6SS-mediated anti-bacterial toxicity in non-pathogenic bacteria. Deciphering the activities of MIX-effectors and how they are secreted will shed light on novel anti-bacterial and virulence mechanisms used by pathogenic bacteria to spread and cause disease. Moreover, it will potentially enable the use of T6SSs and MIX-effectors as future custom-made anti- bacterial agents.